1. Field of the Invention
The present invention relates generally to lipid metabolism. More particularly, the present invention relates to non-radioactive assays for the detection of the lysophospholipase D(LysoPLD) activity by measuring the metabolism of a synthetic substrate analog.
2. Related Art
The role of lipids in cancer development is being recognized as being extremely important and they provide new targets for therapeutics. Many bioactive lysophospholipids, including LPA, LPC, sphingosylphosphorylcholine (SPC), sphingosine 1-phosphate (SIP) and lysophosphatidylserine (lyso-PS) exhibit pleiomorphic effects on multiple cell lineages, including ovarian cancer cells. (Fang, et al., Ann. N.Y. Acad. Sci., 905:188-208. (2000, Luquain, et al., Trends Biochem. Sci., 28:377-83. (2003)) LPA and SIP signal through interaction with specific cell surface receptors of the endothelial cell differentiation gene (edg) family of cell surface seven-transmembrane G-protein coupled receptors (GPCR), whereas SPC and LPC activate the OGR1 family of GPCR. (Hla, et al., Science, 294:1875-8. (2001)) LPA is formed by two routes as shown in FIG. 1, hydrolysis of the sn-2 acyl chain of phosphatidic acid (PA) by a PA-specific PLA2 or by cleavage of the choline head group from lyso-phosphatidylcholine by LysoPLD. The purification, characterization, and identification of the ovarian cancer activating factor (OCAF) from ascites fluid of ovarian cancer patients has demonstrated that OCAF is comprised of numerous forms of LPA, and accounts for the ability of ascites fluid to activate ovarian cancer cells. (Mills, et al., Cancer Res., 48:1066-71. (1988)) LPA, at concentrations present in ascites fluid from ovarian cancer patients (1-80 μM), increases proliferation under anchorage-dependent and -independent conditions, prevents apoptosis and anoikis, increases invasiveness, induces cytoskeletal reorganization and change of shape, decreases sensitivity to cisplatin (the most effective drug in the treatment of ovarian cancer), increases production of vascular endothelial growth factor (VEGF), interleukin-8 (IL8), IL6, urokinase-type plasminogen activator (uPA), and LPA itself, increases activity of matrix metalloprotease-2 (MMP2) and MMP9, and increases mRNA expression for a number of growth factors and other important mediators. (Mills, et al. Cancer Treat. Res., 107:259-83. (2002). Thus LPA plays a critical role in the regulation of proliferation, viability, drug sensitivity, invasiveness and metastasis of ovarian cancer cells, suggesting that it is likely to be an effective target for therapy.
The LPA receptors edg-2, edg-4, and edg-7 are now known as LPA1, LPA2, and LPA3, respectively. Pharmacological concentrations of LPA must be produced extracellularly to induce receptor-dependent biological responses. (Moolenaar, et al., Curr. Opin. Cell Biol., 9:168-73. (1997)) LPA receptors exhibit characteristic responses to LPA species with different chain lengths, different unsaturation patterns, and different acyl positions. (Bandoh, et al., FEBS Lett., 478:159-65. (2000)) In addition, LPA receptors can show marked enantioselectivity as well as isoform-selective activation for a series of 2-substituted N-oleoyl ethanolamide phosphoric acid (NAEPA) analogs. (Heise, et al., Mol. Pharmacol., 60:1173-80. (2001)) For example, LPA produced by stimulated platelets (Moolenaar, et al., Curr. Opin. Cell Biol., 9:168-73. (1997), Moolenaar, Exp. Cell Res., 253:230-8. (1999)) is distinct from the LPA found in ascites fluid from ovarian cancer patients (Mills, et al., J. Natl. Cancer. Inst., 93:1437-9. (2001)), and is an important bioactive lipid in ovarian cancer. (Mills, et al., Cancer Treat. Res., 107:259-83. (2002, Xu, et al., J. Soc. Gynecol. Investig., 8:1-13. (2001)) In platelets, sn-1 LPA is preferentially produced, but ascites fluid contains an elevated amount of sn-2 LPA. (Xu, et al., Clin. Cancer Res., 1:1223-32. (1995)) Lysophospholipase D activity was first characterized over sixteen years ago and recognized as an important source of plasma LPA. (Tokumura, et al., Biochim. Biophys. Acta, 875:31-8. (1986)) At least two forms are known—one which degrades alkyl ether linked LPC (i.e., platelet activating factor, or PAF) which is calcium-dependent and intracellular. The other form, secreted into the plasma, optimally processes acyl-linked LPC but will also hydrolyze PAF and sphingosylphosphocholine.
LysoPLD has an important role in normal physiology. This enzymes activity is increased in normal pregnant women in the third trimester, correlating with the role of LPA in stimulating oocyte maturation and embryonic development. FIG. 1 illustrates the normal route of LPA formation and cell activation, in which conversion of PC to LPC in high-density lipoprotein particles from the liver is coupled to lecithin cholesterol acyltransferase which converts cholesterol (Chol) to cholesteryl esters (CE). (Tokumura, Biochimica et Biophysica Acta—Molecular and Cell Biology of Lipids, 1582:18-25. (2002)) LPC is the second most abundant phospholipid in human serum, with an estimated concentration of 200 μM. (Tokumura, et al., J. Biol. Chem., 277:39436-42. (2002)) LPC can also be produced by the hydrolysis of PC by PLA1 or sPLA2 released from activated cells or tumor cells. The pathophysiology of lysoPLD is also noteworthy. It is upregulated in ascites fluid and serum of patients with ovarian cancer and the LPA thus produced can enhance metastasis of cancer cells through mesothelial cell layers. Imamura, et al., Biochem Biophys Res Commun, 193:497-503. (1993) In addition, it is overproduced in hypercholestermic rabbits causing monocyte activation and attachment to vascular walls, leading to atherosclerosis from increased LPA levels. (Tokumura, et al., J Lipid Res, 43:307-15. (2002)).
Recently, lysoPLD has been purified and cloned. The purified lysoPLD from human serum has increased activity by 14,400-fold, a Km of 260 μM and a Vmax of 103 nmol/min for 16:0 LPC. Similar kinetic parameters were obtained for unpurified human ATX expressed in CHO—K1 cells. LysoPLD/ATX processed LPC more effectively than nucleotides. (Tokumura, et al., J. Biol. Chem., 277:39436-42. (2002)) Comparison with proteins in the database revealed the identity as being ATX, an enzyme previously thought to cleave nucleoside di- and trisphosphates. Cloned rat and human ATX exhibit identical activities to serum lysoPLD.
Autotaxin (ATX) was known as NPP2 and is a 125-kDa glycoprotein that stimulates tumor cell motility and has an in vivo role in tumor progression and invasion. (Nam, et al., Oncogene, 19:241-7. (2000)) ATX/LysoPLD belongs to the nucleotide pyrophosphatase/phosphodiesterase (NPP) family of enzymes which release the nucleoside 5′-phosphate from nucleotide and nucleotide derivatives. Increased ATX mRNA and/or expression levels have been demonstrated in several cancers including ovarian, breast (Yang et al., Clin. Exp. Metastasis, 19:603-608 (2002) hepatocellular (Zhang et al., Chin Med J (Engl) 112: 330-2. (1999)), lung (Yang et al. Am J Respir Cell Mol. Biol. 21:216-22. (1999)), prostate, colon, renal carcinoma (Stassar et al, Br J Cancer, 85:1372-82. (2001)), and Hodgkin's lymphoma.
Recent work has shown that loss of LysoPLD activity in ATX mutants results in a reduction in motogenic ability, directly demonstrating that the production of bioactive phospholipids (i.e. LPA) is important for cell motility. This result suggests that inhibition of increased ATX/LysoPLD activity by small-molecules could be an attractive new avenue for anti-cancer chemotherapy.
Early methods for assaying LysoPLD activity were performed with 14C-palmitoyl-LPC and radio-thin layer chromatography (TLC). This was supplanted by TLC purification of unlabeled LPA with GC analysis of methyl esters, or by measurement of LPA-trimethylsilyl ether by gas chromatography-mass spectrometry (GC-MS). Recently, an enzymatic determination of choline release has been employed. However, none of the existing methods are suited to HTS (high throughput screening) and drug discovery.
Assays using fluorogenic substrates have become widely utilized because of high sensitivity, ease of handling, adaptability for HTS formats, and the ability to follow the kinetics of the reaction without the need for quenching at each time point. Importantly, fluorogenic assays permit continuous monitoring rather than needing be stopped at various endpoints, and they can be adapted from an in vitro biochemical modality to a cell-based in situ assay. One common modality is the conversion of a non-fluorescent substrate, e.g., a fluorescein phospholipid ester, to a fluorescent product. (Zaikova, et al., Bioconjugate Chem., 12:307-13. (2001))
A more complex and versatile approach, employs a “fluorescence dequenching” mode, in which a fluorophore that is “silent” because of intramolecular quenching becomes fluorescent once enzymatic activity liberates it from the intramolecular energy transfer mechanism. Such an approach was demonstrated for PLA2 (Feng, et al., Chemistry & Biology, 9:795-803. (2002)) and the assay for lysoPLD of the present invention (FIG. 2). Other fluorogenic assays have been developed to monitor ceramidase (Nieuwenhuizen, et al., Chem. Phys. Lipids, 114:181-91. (2002)), γ-glutamyl hydrolase, DNA ligase (Sando, et al., J. Am. Chem. Soc., 124:2096-7. (2002, Pankuch, et al., Bioorg. Med. Chem. Lett., 11: 1561-4. (2001)), PLA2 (Hendrickson, et al., Analyt. Biochem., 185:80-3. (1990, Farber, et al., J. Biol. Chem., 274:19338-46. (1999)), and nucleic acid hybridization. (Tyagi, et al., Nat. Biotechnol., 14:303-8. (1996))